The long term goal of this research project is to understand the molecular mechanisms of cellular response to heat stress in mammalian cells. During the next granting period, emphasis will be on the elucidation of molecular control mechanisms that regulate the expression of the 70 kDa heat shock protein hsp7O. We will test a hypothesis inferred from our preliminary studies that there is a dual control mechanism in the heat shock response: a positive control mediated via the heat shock transcription factor HSF1, and a negative control mediated via a novel DNA-binding factor, the constitutive HSE-binding factor (CHBF). The two specific aims and our working hypotheses are as follows: Specific Aim I will test the hypothesis that hsp7O autoregulates its own synthesis by interacting with HSF1, a key positive regulatory protein in the heat shock response. We plan to develop and characterize a model system to study the roles of HSF1 and hsp7O, and interactions between them, in the regulation of heat shock response in mammalian cells. To accomplish this goal we propose: (a) To modify the cellular levels of HSF1, hsp7O, or both at the same time, through the use of retroviral mediated gene transfer technique. Specifically, we plan to generate and characterize rodent cell lines (from Rat-1 cells) that stably and constitutively overexpress (i) human HSF1, (ii) human hsp7O, (iii) both human HSF1 and human hsp7O, and (iv) antisense RNA targeting the constitutive hsc7O and the heat-inducible hsp7O messages; and (b) Using these stable cell lines generated in Specific Aim 1-a, in comparison to control cells, we plan: (i) To examine the cellular response at 37 degrees C, upon heat shock, and during post-heat-shock recovery, and (ii) To examine the biochemical properties of the overexpressed human HSF1 protein, such as the ability of HSF1 to bind to the heat shock element (HSE), the state of oligomerization and phosphorylation of HSF1, and the association of HSF1 with hsp7O or other cellular proteins. Specific Aim II will test the hypothesis that CHBF may function as a negative regulatory factor in the heat shock response, such as by repressing heat-induced hsp7O expression. To accomplish this goal we propose: (a) To purify CHBF from mammalian cells to apparent homogeneity by column chromatography, including the use of HSE-oligonucleotide affinity column, (b) To identify the protein, and to examine the possibility that CHBF may be identical or closely related to a protein known as Ku-autoantigen, (c) To clone and sequence the gene encoding the rat CHBF, and (d) to develop and characterize a model system to study the roles of CHBF in the regulation of heat shock response. We plan: (i) To generate rodent cell lines that stably and constitutively overexpress CHBF, and to examine their response at 37 degrees C, upon heat shock and during post-heat-shock recovery, and (ii) To map the distribution of CHBF on DNA (specifically the hsp7O promoter sequence) in cells by UV- crosslinking technique. Results from this study will assist us in understanding the functions of HSF1 and CHBF, and their involvement in the dual control mechanism of heat shock gene expression.